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J. Med. Chem. 2005, 48, 5604-5607
Antitumor Activity of New Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and Study of Their Effect on the Cell Cycle1 Aldo Andreani,*,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,† Mirella Rambaldi,† Vida Garaliene,‡ William Welsh,§ Sonia Arora,§ Giovanna Farruggia,| and Lanfranco Masotti| Dipartimento di Scienze Farmaceutiche, Universita´ di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Lithuanian Institute of Cardiology, Kaunas, Lithuania LT-3007, Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey (UMDNJ), 675 Hoes Lane, Piscataway, New Jersey 08854, and Dipartimento di Biochimica “G. Moruzzi”, Universita´ di Bologna, Via Irnerio 48, 40126 Bologna, Italy Received April 15, 2005
This paper reports the synthesis of a new series of 3-(5-imidazo[2,1-b]thiazolylmethylene)-2indolinones which were tested as potential antitumor agents at the National Cancer Institute. Two derivatives are now under review by BEC (Biological Evaluation Committee of NCI). To investigate the mechanism of action, the effect on cell cycle progression was studied by monitoring them in colon adenocarcinoma HT-29: both were able to block HT-29 in mitosis. 3-[(2,6-Dimethylimidazo[2,1-b]thiazol-5-yl)methylene]-5-chloro-2-indolinone was the most active compound. Introduction In 1997 we published a paper on the antitumor activity of 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones. The compounds reported in that paper2 were mainly formed by connecting an imidazo[2,1-b]thiazole moiety (bearing at the 6 position Cl, CH3 or C6H5) by means of a methine bridge to 2-indolinone or its 5-methoxy analogue. The promising results obtained prompted us to prepare a new series of analogues including, as starting materials, several indolinones and imidazothiazoles different at positions 2 and 3. The study3 confirmed that the most potent compounds bear a methoxy group at the 5 position of the indole system; moreover, in the imidazothiazole portion the substitution at the 2 position is much more important than at the 6 position, since all the most interesting compounds are 2-methyl derivatives. In this paper we describe new analogues with substitutions in the indole as well in the imidazothiazole portion. The antitumor activity of all new compounds was evaluated on three human tumor cell lines according to the protocols available at the National Cancer Institute (NCI, Bethesda, MD), and the active compounds were tested on the sixty tumor cell lines as well. Since it is well-known that several indole derivatives can induce G2/M arrest4,5 and apoptosis in certain cell lines,6 the effect on cell proliferation and cell cycle progression in the colon adenocarcinoma cell line HT29 was also studied for the most potent antitumor derivatives. Finally, they were tested as positive inotropic agents in a search for potential coanthracyclinic activity.7 * Corresponding author: tel +39-051-2099714, fax +39-051-2099734, e-mail
[email protected]. † Universita ´ di Bologna. ‡ Lithuanian Institute of Cardiology. § University of Medicine & Dentistry of New Jersey. | Universita ´ di Bologna.
Chemistry. The substitutions (Scheme 1 and Table 1) have been performed according to the following rationale: (1) in order to understand the influence of the substituents at the 5 and 6 positions of the indole system, we condensed two imidazothiazole aldehydes (5, 6) with four different indolinones (10-13). This reaction gave compounds 16-23; (2) compound 24 was synthesized in order to study the effect of the N-substitution in the indole moiety of the most active compound of the previous series;2 (3) new substituents have been introduced in the imidazothiazole moiety while maintaining the same indolinone: chlorine at the 2 position (25 and 26) and trifluoromethyl at the 6 position (27); (4) finally, a single compound was prepared (28) with the shift of the substituent from the 2 to the 3 position of the imidazothiazole. Compounds 16-28 were prepared by means of the Knoevenagel reaction between the aldehydes 4-9 and the indolinones 10-15 in the presence of piperidine. The aldehyde 4 (starting compound for the synthesis of 27) was obtained by means of the Vilsmeier reaction on the corresponding imidazo[2,1-b]thiazole 3, prepared in turn from 2-amino-5-methylthiazole 1 and 1-bromo-3,3,3trifluoroacetone. The intermediate compound 2 was isolated and used in the subsequent step without further purification. The structures of the final compounds (1628) were confirmed by means of IR and 1H NMR spectra (see Supporting Information). They were obtained as pure geometrical isomers which, according to the usual NOE experiments described in the previous papers,2,3 were assigned to the E configuration. Results and Discussion (a) Antitumor Activity (in vitro growth inhibition and cytotoxicity). As a preliminary screening, compounds 16-28 were evaluated for their cytotoxic potency on three human cell lines: NCI-H460 lung cancer, MCF7 breast cancer, and SF-268 glioma. A
10.1021/jm050353e CCC: $30.25 © 2005 American Chemical Society Published on Web 07/28/2005
Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5605
Scheme 1
Table 1. Compounds 16-28 compd
x-y
R
R1
R2
R3
formula
MW
mp, °C
16 17 18 19 20 21 22 23 24 25 26 27 28
CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH ClCdCH ClCdCH CH3CdCH HCdCCH3
Cl Cl Cl Cl CH3 CH3 CH3 CH3 CH3 Cl CH3 CF3 CH3
H H H H H H H H CH3 H H H H
Cl OH OH OCH3 Cl OH OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
H H CH3 CH3 H H CH3 CH3 H H H H H
C15H9Cl2N3OS C15H10ClN3O2S C16H12ClN3O2S C17H14ClN3O2S C16H12ClN3OS C16H13N3O2S C17H15N3O2S C18H17N3O2S C18H17N3O2S C15H9Cl2N3O2S C16H12ClN3O2S C17H12F3N3O2S C17H15N3O2S
350.2 331.4 345.8 359.4 329.8 311.3 325.4 339.4 339.4 366.2 345.8 379.4 325.4
268-270 dec 288-290 dec 300-303 dec 289-290 dec 295-298 dec 300-305 dec 310-313 dec 264-266 dec 163-168 265-267 dec 231-233 260-263 255-257
Table 2. Sixty Cell Line Panel (growth inhibition and cytostatic and cytotoxic activity of the selected compounds) NSC
compda
725098
20
725100
21
725102 725099
23 24
67574
vincristine sulfate c
modes leukemia NSCLC GI50 TGI LC50 GI50 TGI LC50 GI50 GI50 TGI LC50 GI50 TGI LC50
-7.70 -6.28 -5.11 -4.47 -4.47 -5.64 -7.00 -4.80 -3.20
-6.17 -4.65 -4.16 -5.40 -4.36 -4.02 -4.24 -5.11 -4.40 -6.60 -4.80 -3.60
colon
CNS
melanoma ovarian
renal
prostate breast MG-MIDb
-6.72 -5.06 -4.34 -5.78 -4.35 -4.03 -4.21 -5.49 -4.55 -4.09 -7.00 -5.40 -4.10
-6.13 -5.08 -4.16 -5.41 -4.50 -4.02 -4.08 -5.46 -4.59 -6.90 -5.20 -3.70
-6.21 -4.60 -4.06 -5.41 -4.50 -4.08 -4.22 -5.26 -4.45 -4.13 -6.80 -5.10 -3.60
-6.54 -4.85 -4.06 -5.30 -4.32 -4.09 -5.32 -4.40 -6.50 -4.70 -3.60
-6.98 -5.62 -4.36 -5.52 -4.42 -4.23 -5.06 -4.10 -6.90 -5.20 -3.50
-5.91 -4.82 -4.30 -5.16 -4.09 -4.12 -5.16 -4.42 -4.02 -6.50 -4.70 -3.50
-6.51 -4.85 -4.02 -5.56 -4.66 -4.33 -5.60 -4.87 -4.03 -6.50 -5.10 -3.50
-6.42 -4.81 -4.15 -5.48 -4.44 -4.05 -4.22 -5.35 -4.46 -4.03 -6.70 -5.00 -3.60
a All values are log. Highest concentration ) 10-4 M; only modes showing values 97% pure) was reconstituted to 3 mg/mL in G-PEM buffer (80 mM PIPES pH 6.8, 0.5 mM EGTA, 2.0 mM MgCl2, 1.0 mM GTP, and 5% glycerol). Polymerization reaction was initiated by mixing 100 µL of reconstituted tubulin with vehicle or test compound (10 µM final concentration) into each well of the prewarmed plate. Polymerization kinetics were then measured at 37 °C for 1 h at 340 nm using a TECAN GENiospro microplate reader (TECAN U.S. Inc., Research Triangle Park, NC). (d) Positive Inotropic Activity. The experiments were carried out on the guinea-pig papillary muscles according to a procedure previously described.7
Acknowledgment. This work has been supported by a grant from MIUR-COFIN 2002, contract number 2002033121_003. We are grateful to the National Cancer Institute (Bethesda, MD) for the antitumor tests. Supporting Information Available: Additional data on biological effects (Figure 1S: percentage of cell death. Figure 2S: time-dependent antiproliferative effect of compound 20 and 21. Figure 3S: cytograms of BrdU incorporation versus PI fluorescence. Figure 4S: cell cycle phase distribution at different times). C, H, N analytical data. IR and 1H NMR spectroscopic data. This material is available free of charge via Internet at http://pubs.acs.org.
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